Vaccine-induced cytokine responses in a guinea pig model of pulmonary tuberculosis.

Guinea pigs exposed to very small numbers of virulent tubercle bacilli by the respiratory route develop a disease which mimics many of the important features of the pathogenesis of human tuberculosis (TB), including the expression of strong protective immunity following vaccination with BCG. In order to elucidate the precise immunological mechanisms of vaccine-induced resistance in this model, both mRNA and protein assays for several guinea pig cytokines and chemokines have been developed. The coordinated expression of cytokine and chemokine mRNA and protein was examined in various leukocyte populations and in inflammatory cells and fluid collected following the induction of tuberculous pleurisy in BCG-vaccinated guinea pigs. Real-time RT-PCR assays revealed that the mRNA levels for IFNgamma, TNFalpha, and IL-8 rose over the first few days of TB pleuritis and then declined over the 9 days of the study. Injection of anti-TGFbeta on day 8 following pleurisy induction resulted in significant changes in cytokine mRNA levels and PPD-induced proliferation in pleural effusion lymphocytes taken 24h later. BCG vaccination induced significantly higher levels of bioactive TNFalpha protein in the supernatants of alveolar, peritoneal and splenic cells from BCG-vaccinated guinea pigs cultured in the presence of attenuated or virulent mycobacteria. In sharp contrast, following virulent challenge, all three cell types from BCG-vaccinated guinea pigs produced significantly less TNFalpha. Thus, BCG vaccination appears to modulate the potentially harmful effects of TNFalpha in this model of pulmonary TB. Levels of mRNA for IL-12p40 were upregulated by exposure of infected and uninfected macrophages to recombinant guinea pig (rgp)TNFalpha. The intracellular survival of mycobacteria was enhanced when endogeous TNFalpha activity was neutralized with anti-rgpTNFalpha antiserum. rgp RANTES (CCL5) upregulated mRNA levels for TNFalpha, IL-1beta, MCP-1 (CCL2), and IL-8 (CXCL8) in alveolar and peritoneal macrophages. These results illustrate the profound effects of prior vaccination with BCG on the cytokine and chemokine responses of distinct cell populations in the guinea pig following exposure to attenuated and virulent strains of M. tuberculosis.

Effects of transdermal nicotine on craving, withdrawal and premenstrual symptomatology in short-term smoking abstinence during different phases of the menstrual cycle.

Gender differences in nicotine response with regard to nicotine sensitivity and withdrawal symptomatology have been reported, with the suggestion that ovarian hormones play a role. Few studies, however, have directly assessed hormonal influences on nicotine response. This study focused on the effects of a transdermal nicotine patch in women during acute smoking abstinence when tested in different phases of their menstrual cycle. Thirty women were randomized to order of menstrual cycle phase (late luteal or follicular) and patch condition (active or placebo). Two 7-day outpatient-testing periods were conducted with 2 days of ad lib baseline smoking and 5 days of smoking abstinence. Dependent measures included scores from the Minnesota Nicotine Withdrawal Scale, Questionnaire on Smoking Urges, and Premenstrual Assessment Form, as well as weight. The severity of both premenstrual symptomatology and nicotine withdrawal symptoms was greater in the late luteal phase. Correlation coefficients confirmed overlap between premenstrual and withdrawal symptomatology, especially for the affect subscale. A significant patch effect was observed, showing diminished craving and premenstrual affect on pain subscale scores for women on active patch. Results showed that nicotine craving and premenstrual pain and water retention symptoms were diminished in women on transdermal patch, and that this effect was greatest in the late luteal phase. In addition, the greatest weight gain was demonstrated for participants in the late luteal phase, placebo condition. In summary, during short-term smoking abstinence in women, transdermal nicotine appears to have a more pronounced effect in the late luteal phase than in the follicular phase in reducing craving and certain premenstrual symptoms.

Energy intake and energy expenditure during the menstrual cycle in short-term smoking cessation.

The effect of short-term smoking abstinence on energy intake and expenditure parameters was investigated for women in different phases of the menstrual cycle (follicular or late luteal) in a rigorous inpatient laboratory setting. Twenty-one participants were randomized to a continued smoking (n = 5) or a smoking abstinence (n = 16) group and admitted for 2 7-day inpatient periods during alternate cycle phases. The smoking abstinence group experienced 2 days of baseline smoking and 5 days of smoking abstinence. Measurements included caloric intake (kcal/24 hours), energy expenditure (by indirect calorimetry), and weight. Results of within-subject analyses indicated no smoking abstinence effect on mean daily total kilocalorie intake, sweet kilocalorie intake, or resting metabolic rate. However, a significant cycle phase effect was observed, with increased kilocalorie intake and expenditure-as well as minor weight gain-occurring during the late luteal phase when premenstrual symptoms are highest. In light of this phase effect, women smokers might benefit by attempting to quit smoking during the follicular phase of their cycle.

Cloning, sequencing, chromosomal location, and function of cDNAs encoding an opioid growth factor receptor (OGFr) in humans.

The native opioid growth factor (OGF), [Met(5)]-enkephalin, is a tonic inhibitory peptide that modulates cell proliferation and tissue organization during development, cancer, cellular renewal, wound healing, and angiogenesis. OGF action is mediated by a receptor mechanism. We have cloned and sequenced cDNAs encoding multiple spliced forms of a human OGF receptor. The open reading frame in the longest cDNA was found to encode a protein of 697 amino acids, and 8 imperfect repeats of 20 amino acids each were a prominent feature. Altogether, five alternatively spliced forms were observed. The cDNA hybridized to mRNA from a variety of normal and neoplastic cells and tissues. Functional studies using antisense oligonucleotides to OGFr demonstrated an enhancement in cell growth. Fluorescent in situ hybridization (FISH) experiments showed the chromosomal location to be 20q13.3. This OGF receptor has no homology to classical opioid receptors. These results provide molecular validity for the interaction of OGF and OGF receptor in the regulation of growth processes in humans.

Cloning, sequencing, expression and function of a cDNA encoding a receptor for the opioid growth factor, [Met(5)]enkephalin.

The native opioid growth factor (OGF), [Met(5)]enkephalin, is a tonic inhibitory peptide that modulates cell proliferation and tissue organization during development, cancer, cellular renewal, wound healing and angiogenesis. OGF action is mediated by a receptor mechanism. We have cloned and sequenced a 2.1-kilobase (kb) cDNA for a receptor to OGF (OGFr). The open reading frame was found to encode a protein of 580 amino acids, and eight imperfect repeats of nine amino acids each were a prominent feature. The protein encoded by this cDNA exhibited the pharmacological, temporal and spatial characteristics of the OGFr. Functional studies using antisense technology demonstrated an enhancement in cell growth. The molecular organization of the OGFr has no homology to classical opioid receptors. These results provide molecular validity for the interaction of OGF and OGFr in the regulation of growth processes.

Withdrawal and pre-menstrual symptomatology during the menstrual cycle in short-term smoking abstinence: effects of menstrual cycle on smoking abstinence.

This study employs a rigorous inpatient laboratory setting to test the hypothesis that withdrawal symptomatology in short-term smoking cessation in women is increased in the late luteal phase when pre-menstrual symptomatology is the highest. Twenty-one female smokers with clinical, anatomical, and hormonal verification of their menstrual cycle phase were randomized to either a smoking abstinence group (n = 16) or a continued smoking group (n = 5). Participants were admitted to the General Clinical Research Center during alternate phases of their cycle for two 7-day admissions with a 1-month interim period when they resumed smoking. Dependent measures, i.e., Minnesota Nicotine Withdrawal Scale scores, Questionnaire on Smoking Urges scores and Pre-menstrual Assessment Form scores were collected during 2 days of baseline and 5 days of smoking deprivation. Smoking behavior was documented by self-report, breath CO levels and saliva cotinine measurements. Withdrawal symptomatology was not affected by menstrual cycle phase during short-term cessation in spite of increased pre-menstrual changes seen in the late luteal phase. In addition, no phase effect on smoking behavior was detected and cigarette consumption remained stable across the cycle in both groups. These results suggest that for some smoking cessation studies, complex strategies to control for menstrual cycle effects may not be necessary. However, Smoking Urges scores did suggest increased desire to smoke and desire to relieve negative affect in the late luteal phase when women have higher pre-menstrual symptomatology. This suggests women may have greater difficulty quitting smoking in late luteal phase, and it seems prudent to recommend that women quit during the follicular phase of their cycle.

Cotinine: effects with and without nicotine.

The purpose of this study was to examine the effects of the metabolite of nicotine, cotinine, in comparison to the effects of the nicotine patch, and a combination thereof during cigarette abstinence. More specifically, this study examined the effects of cotinine on physiological measures, subjective measures assessing craving, withdrawal symptoms and mood, and performance measures. A between-subject, 2 x 2 factorial design was used, with the daily administration of a 15-mg nicotine patch (Nicotrol) versus placebo patch as one factor and 80 mg of oral cotinine fumarate versus placebo drug as the other factor. Baseline measures were obtained while the subjects smoked cigarettes on an ad lib basis for 1 week. Subjects (n = 106) were then randomly assigned to one of four treatment conditions and for the next 14 days were required to be abstinent from cigarettes and take the study drugs. Cotinine administration, with or without nicotine patch, produced serum cotinine concentrations 3 4 times higher than during ad lib smoking. Results showed a reduction of self-reported tobacco withdrawal symptoms using the nicotine patch alone. Cotinine alone had no effect on withdrawal symptoms. However, when nicotine patch was combined with cotinine, the beneficial effect of the nicotine patch on withdrawal symptoms was absent. Therefore, cotinine appears to antagonize the effects of nicotine in the alleviation of withdrawal symptoms at concentrations higher than that attained from normal smoking. This effect does not appear to be mediated by changes in nicotine disposition.

Safety of single-dose administration of an adeno-associated virus (AAV)-CFTR vector in the primate lung.

Gene therapy for cystic fibrosis (CF) would ideally be accomplished with a vector capable of long-term expression of the cystic fibrosis transmembrane conductance regulator (CFTR) in the absence of a host inflammatory response. Recombinant adeno-associated virus (AAV)-CFTR vectors possess these characteristics in rabbits. Because the utility of AAV vectors as gene transfer agents has only been recognized recently, AAV vector-mediated transduction has never been modeled in a primate host, which is an important step before its use in humans. In order to test the safety and biological activity of AAV-CFTR, single doses of AAV-CFTR vector were administered by fiberoptic bronchoscopy to the posterior basal segment of the right lower lobe (RLL) of the lungs of 10 rhesus macaques with four matched vehicle-treated controls. Animals were followed for 10, 21, 90 or 180 days following vector instillation. Vector DNA transfer occurred in bronchial epithelial cells in the RLL of each animal that received vector as assessed by in situ DNA PCR. Vector mRNA was detectable for 180 days after administration as detected by RT-PCR and by RNase protection assay. Safety of vector administration was determined by measurements of pulmonary mechanics, arterial blood gas analysis, chest radiographs, and bronchoalveolar lavage (BAL) fluid analysis including cell count and quantification of inflammatory cytokines. Gross and microscopic pathologic examination were also performed. There was no evidence of inflammation or other toxicity, although vector DNA was found in extrapulmonary organs of some animals. These results indicate that transduction of the primate airway epithelium with the AAV-CFTR mediates long-term CFTR cDNA transfer and is relatively safe.

Symptomatology and energy intake during the menstrual cycle in smoking women.

This study extends our understanding of smoking behavior, withdrawal, and premenstrual symptomatology in smoking women, as well as energy intake as a function of cycle phase. Thirty-two women age 18 to 40 years, smoking ad lib, were followed for an average of two menstrual cycles with hormonal verification. Withdrawal and premenstrual symptomatology as well as energy intake were reported during the follicular (F), luteal (L), and late luteal (LL) cycle phases. Both premenstrual symptomatology and withdrawal symptomatology were higher during the LL phase. Subject response on measurements of craving, irritability, restlessness, increased appetite, and depressed mood tended to be higher in the LL phase. Energy intake did not vary as a function of cycle phase. Participants perceived that they smoked more and had increased appetite in the LL phase but related measurements did not confirm this. Premenstrual and withdrawal symptoms are highly correlated and one needs to be cautious in interpreting cycle effects on withdrawal. Overall symptomatology seems to be lower during the F phase, indicating that this may be a more opportune time for women to quit smoking.

Effects of treatment on cardiovascular risk among smokeless tobacco users.

BACKGROUND: Studies show sustained levels of nicotine among young males using smokeless tobacco, causing concern for subsequent cardiovascular risk. Also, there is little information on effects of nicotine replacement on cardiovascular risk in cessation programs. This study investigates the effects of nicotine gum replacement in smokeless tobacco cessation on cardiovascular risk factors. METHODS: Smokeless tobacco users, ages 18-65, were randomly assigned in a double-blind fashion to 2-mg nicotine or placebo gum. At baseline, Week 4, and Week 8, dependent measurements, heart rate, blood pressure, and weight were recorded, and fasting lipoprotein profiles were drawn. RESULTS: This paper focuses on the smokeless tobacco users who refrained from use during the study period (N = 56). The nicotine gum group weighed less (P = 0.033) than the placebo group throughout the study and weight increased at a significant rate between Weeks 4 and 8 for both groups as gum decreased. Triglycerides were higher for the nicotine gum group than the placebo group (P = 0.031), with triglycerides decreasing between Weeks 4 and 8, with a similar effect seen among nonabstinent smokeless tobacco users. There was no dose, time, or dose by time effect for the other dependent measures. CONCLUSIONS: Among smokeless tobacco users who were abstinent, weight increased, with subjects on nicotine gum weighing less throughout the study. The lipoprotein profile, heart rate, and blood pressure did not improve over time, contrary to smokers in whom HDL increases and heart rate decreases with cessation. This could relate to different routes of administration, pharmacokinetics, or by-products of tobacco smoking being absent in smokeless tobacco. In addition, nicotine gum appeared to have neither an adverse nor a positive effect on heart rate, blood pressure, LDL, HDL, or total cholesterol.

CFTR regulates outwardly rectifying chloride channels through an autocrine mechanism involving ATP.

The cystic fibrosis transmembrane conductance regulator (CFTR) functions to regulate both Cl- and Na+ conductive pathways; however, the cellular mechanisms whereby CFTR acts as a conductance regulator are unknown. CFTR and outwardly rectifying Cl- channels (ORCCs) are distinct channels but are linked functionally via an unknown regulatory mechanism. We present results from whole-cell and single-channel patch-clamp recordings, short-circuit current recordings, and [gamma-32P]ATP release assays of normal, CF, and wild-type or mutant CFTR-transfected CF airway cultured epithelial cells wherein CFTR regulates ORCCs by triggering the transport of the potent agonist, ATP, out of the cell. Once released, ATP stimulates ORCCs through a P2U purinergic receptor-dependent signaling mechanism. Our results suggest that CFTR functions to regulate other Cl- secretory pathways in addition to itself conducting Cl-.

Alternate translation initiation codons can create functional forms of cystic fibrosis transmembrane conductance regulator.

To evaluate the function of transmembrane domain 1 (TMD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) and the methionines that function in translation initiation, a series of progressive 5' truncations in TMD1 were created to coincide with residues that might serve as translation initiation codons. Expression of the mutants in Xenopus oocytes demonstrated that internal sites in TMD1 can function as initiation codons. In addition, all of the mutants that progressively removed the first four transmembrane segments (M1-M4) of TMD1 expressed functional cAMP-regulated Cl- channels with ion selectivity identical to wild-type CFTR but with reduced open probability and single channel conductance. Further removal of transmembrane segments did not produce functional Cl- channels. These data suggest that segments M1-M4 are not essential components of the conduction pore or the selectivity filter of CFTR.

Cholesterol changes in smoking cessation using the transdermal nicotine system. Transdermal Nicotine Study Group.

BACKGROUND: Cigarette smoking is a well-known major risk factor in coronary heart disease. Smoking cessation results in a positive change in atherogenic factors. High-density lipoprotein cholesterol has been observed as increasing with smoking cessation. Since the use of nicotine transdermal replacement has become so widespread, this study examined the effect, if any, of the transdermal nicotine system on selected cardiovascular parameters in patients who were abstinent from cigarette smoking, and possible dose effect. METHODS: Eight cardiovascular outcome measures were evaluated at baseline and Week 6 in both abstinent and non-abstinent patients randomized to four treatment groups; transdermal nicotine system 7 mg, 14 mg, and 21 mg per day, and placebos. RESULTS: In abstinent patients, systolic blood pressure and heart rate decreased from baseline (while still smoking, before the start of the study) to the end of transdermal treatment, while weight increased. Similarly, HDL increased while LDL decreased and triglycerides increased. In nonabstinent patients, weight also increased from baseline to Week 6 while heart rate decreased. No other variables showed significant change. In abstinent patients, effect of nicotine dosage was observed with greater weight gain in placebo than 21 mg TTS patients and greater decrease in heart rate in placebo than 21 mg TTS patients. CONCLUSIONS: In summary, the abstinent patients showed a positive effect of smoking cessation on cardiovascular risk factors even while using the transdermal nicotine system. These findings are favorable since the transdermal nicotine system has become a useful method of nicotine replacement in smoking cessation programs.

A comparison of knowledge of medical students and practicing primary care physicians about cardiovascular risk assessment and intervention.

BACKGROUND: Heart attack and stroke are still prevalent causes of death and disability in the U.S. adult population (1, 2). Studies (3-9) have shown that modification of hypertension, smoking, and hypercholesterolemia can reduce risks for atherosclerosis and subsequent cardiovascular events. Therefore, it is important that physicians be skilled in assessing and modifying patients' overall cardiovascular risk. This study compares acquired knowledge of second-year medical students about cardiovascular risk assessment with knowledge in a selected group of practicing primary care physicians, who are members of the medical school's clinical faculty, using a new experimental testing technique called the tailored response test (TRT). METHODS: Students performed a structured cardiovascular risk intervention on a patient in primary care clinics. Their acquired knowledge was then tested using the TRT, which contained 43 discrete judgments about a clinical case. Test scores of students and faculty were compared. RESULTS: Both students and faculty demonstrated knowledge about the most important risk factors, appropriate screening tools, and interventions. However, the selected physicians did not demonstrate knowledge of certain important risk assessment and intervention recommendations, based on national standards. Only 38% of faculty and 27% of students were aware that a "fasting" serum cholesterol is not needed for screening, 30% of faculty believed that if cholesterol was over 300 they would "probably prescribe medicine" before other intervention strategies were tried, and 32% of faculty and 30% of students would order a screening chest X-ray, which is incorrect in the case history. CONCLUSIONS: The TRT, in contrast to self-report surveys, demonstrates that important cardiovascular risk assessment and intervention knowledge, with implications for cost effectiveness in health care delivery, has not penetrated to a selected group of physicians who are members of the medical school's clinical faculty and therefore serve as role models for medical students. This is disturbing, in light of current emphases on cost effectiveness in health care. Greater undergraduate curricula and CME emphasis on cardiovascular preventive practice is needed, such that almost 100% of students and faculty demonstrate knowledge, and practice, of preventive medicine according to national standards. In turn, groups developing national standards are enjoined to design and implement effective approaches for disseminating these recommendations.

Patient perceptions about the influence of cholesterol on heart disease.

National surveys have shown that adults need to known more about their own blood cholesterol levels and about specific methods for lowering blood cholesterol. We examine patient perceptions of cholesterol and heart disease after their participation in a cholesterol management program designed for the primary care setting. We counseled 221 patients and successfully interviewed 179 by telephone four weeks after their initial dietary counseling visit. Nine of 10 patients could report their cholesterol level, and 98% of patients could appropriately label it as borderline-high or high-risk. Ninety-seven percent of the patients could give one valid reason why cholesterol was important to their health. Many patients knew specific dietary recommendations from counselors. Patients positively reviewed counseling and the cholesterol management program. A large percentage of patients had already made changes in diet and eating habits at the time of the interview. Total cholesterol measurements showed a mean reduction of 8.5% postcounseling.

The shortened premenstrual assessment form.

Research on the premenstrual syndrome (PMS) has been impeded by a lack of reliable and valid assessment techniques. The premenstrual assessment form (PAF), although a valid and reliable instrument, consists of 95 questions, requires extensive periods of time to complete and may be inappropriate for some clinical and research purposes. A study was designed to shorten the PAF and to test the validity and reliability of the shortened instrument. Twenty items that were most frequently reported to change during the week prior to menses were selected from the 95-item PAF form. The 20-item PAF form was administered at the baseline and 6- and 12-month follow-up clinic visits. A factor analysis identified three subscales: affect, water retention and pain. Our results showed that a shortened, 10-item version of the PAF had high internal consistency and reliability. A comparison of the symptoms on the 10-item PAF scale to reported nicotine withdrawal symptoms indicated that while the two correlated, the intercorrelation between the PAF subscales and the total PAF over time was higher. Thus, the 10-item PAF appears to measure a somewhat distinct and relatively stable set of symptoms. The 10-item PAF is a reliable and valid instrument that can be used to assess PMS when the study design or clinical need precludes the use of a 95-item PAF.

Structured clinical teaching strategy.

This study investigated the impact of a structured exercise on low back pain, as part of a second year ambulatory course, on students' low back pain examination skills. One-hundred and eighty-eight medical students participated in one of four types of instructional intervention: 1) structured clinical exercise and reading, 2) random clinical experience and no reading, 3) reading only, and 4) no clinical experience or reading. At the end of the year, students completed an Objective Structured Clinical Exam (OSCE) in which two stations assessed back pain history and physical exam skills. An analysis of variance of the OSCE scores showed no significant difference in students' performance in relation to the type of instructional intervention. The General Professional Education of the Physician Report argues for the importance of structured clinical education. In medical education, unpredictable patient exposure and crowded curricula frequently result in students having none or only one structured learning opportunity to acquire critical skills. Unfortunately, this study found that assuring at least one structured clinical experience for a specific common problem seen in ambulatory care did not enhance student ability to select and use specific history or physical exam skills for that problem as assessed by an OSCE, as compared with students who did not have this experience. To assure that essential clinical skills are acquired, it most likely requires that both systematic instructional strategies and repeated learning opportunities are available to reinforce learning.

A mini-workshop to train medical students to use a patient-centered approach to smoking cessation.

Leaders in preventive medicine and medical education have called for more attention to preventive medicine in medical education curricula. This study describes the implementation of a training program designed to introduce preventive medicine skills into the medical school curriculum. The specific issue addressed was smoking cessation. A two-hour workshop on the patient-centered approach to smoking intervention was presented to medical students during the family medicine rotation of the second-year clinical medicine course sequence. Two of the four student groups in the family medicine clinical rotation received the training and were afforded practice opportunity with at least one smoking patient at the clinical site. The other two groups went through the usual rotation with no special instruction or clinical emphasis on preventive interventions. Second-year medical students expressed positive perceptions of preventive medicine as assessed by self-rating on attributes important to successful preventive practice. These positive perceptions were retained by both groups after the clinical experience. Students with the workshop training were more confident in their smoking intervention skills and performed better overall on an objective clinical evaluation of intervention skills.